FoxO1a Can Alter Cell Cycle Progression by Regulating the Nuclear ...

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Molecular Endocrinology 18(7):1756–1767 Copyright © 2004 by The Endocrine Society doi: 10.1210/me.2004-0071

FoxO1a Can Alter Cell Cycle Progression by Regulating the Nuclear Localization of p27kip in Granulosa Cells MELISSA A. CUNNINGHAM, QIN ZHU,

AND

JAMES M. HAMMOND

Department of Medicine, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Forkhead transcription factors of the FOXO family are important downstream targets of the phosphatidylinositol 3-kinase pathway, which has been shown to play a critical role in cell proliferation and cell survival. Activation of FOXOs can block cellular proliferation and drive cells into a quiescent state. In certain cell types, this cell cycle arrest is dependent on the transcriptional induction of the cellcycle inhibitor p27kip. In granulosa cells, which go through an exponential growth phase during development of the ovarian follicle, we find that FoxO1a is a key regulator of the G1/S transition in these cells. Overexpression of a dominant-negative version of FoxO1a (Foxo1a-⌬256; a C-terminal truncation mutant that possesses a functional

DNA-binding domain, but lacks a transactivation domain) causes a dramatic increase in S-phase cells (>8-fold increase by both DNA content and bromodeoxyuridine incorporation assays). Surprisingly, this is not dependent on transactivation of the p27kip gene. We provide evidence that when FoxO1a activity is impeded, p27kip protein is largely localized to the cytosol, suggesting that FoxO1a blocks cell cycle entry by altering the compartmentalization of p27kip within the cell, increasing its concentration in the nucleus. These studies demonstrate for the first time that FoxO1a can regulate p27kip nuclear localization. (Molecular Endocrinology 18: 1756–1767, 2004)

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regulators of follicular growth and development and prompted us to investigate the actions and importance of FoxO1a in a well-characterized granulosa cell culture system (12). In other cell types, the FOXO subfamily of forkhead transcription factors transactivates a number of genes that regulate cell proliferation and survival (13–15). Growth factor activation of the PI3-kinase pathway is thought to promote proliferation and survival in part by causing phosphorylation and inactivation of FOXO family members. The FOXO factors induce genes such as the cyclin-dependent kinase (cdk) inhibitor p27kip and Fas ligand, which cause cell-cycle arrest and/or apoptosis, respectively (16–18). Overexpressing FOXO transcription factors strongly inhibits cell proliferation in a variety of cell types (16, 18–20) causing arrest in the G1 phase of the cell cycle (20–23). This FOXO-induced cell cycle arrest effect is, in part, due to an up-regulation of p27kip (16, 20). Increased levels of p27kip cause G0/G1 arrest by inhibiting cyclin/cdk complexes necessary for S-phase entry and progression. In some cells, induction of p27kip by FOXO factors is a result of direct transcriptional activation of the p27kip gene (16, 24, 25). However, FOXO1 may also regulate p27kip posttranscriptionally by prolonging its half-life (20). In addition to the tight control of p27kip abundance, there is also important regulation at the level of p27kip subcellular localization. Throughout the cell cycle, p27kip shuttles between the nucleus and cytoplasm, with its import/export regulated by specific phosphor-

N THE OVARY, the cyclical growth of the follicle(s) selected for ovulation is accompanied by exponential multiplication of granulosa cells. This replication is known to be dependent upon FSH and IGF-I in vivo (1), and knockout studies of either of these factors in mice results in early arrest of follicle growth and infertility (2, 3). In recent years, the phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway has been implicated in the proliferation and survival of ovarian cells in response to both growth factor and gonadotropin stimulation (4–7). However, the downstream mediators of this pathway remain largely unknown. Data from our lab and another have demonstrated that FoxO family members [FOXO-human; Foxo-murine; FoxO-other species (8)] are highly expressed and regulated in the ovary and are targets of FSH and IGF-I via the PI3kinase pathway (9, 10). Recently, it was demonstrated that knockout of Foxo3a causes premature initiation of ovarian follicular growth which, in turn, leads to early depletion of follicles and premature ovarian failure (11). These data suggested that Foxo factors are critical Abbreviations: BrdU, Bromodeoxyuridine; cdk, cyclin-dependent kinase; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HA, hemagglutin; PCNA, proliferating cell nuclear antigen; PI, propidium iodide; PI3-kinase, phosphatidylinositol 3-kinase; WT, wild-type. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

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Cunningham et al. • FoxO1a Can Regulate p27kip Nuclear Localization

Mol Endocrinol, July 2004, 18(7):1756–1767

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ylation events (26–30). For example, p27kip is almost exclusively nuclear during G0, but when cells are stimulated by growth factors or other mitogens, p27kip is phosphorylated on Ser10 and exported to the cytoplasm (26). In the ovary, p27kip is hormonally regulated during follicular development and is expressed to the greatest extent in granulosa cells undergoing terminal differentiation or in fully differentiated luteal cells (31). Foxo1a is also expressed and hormonally regulated in the ovary (9, 10). In cultured granulosa cells, its phosphorylation state and subcellular localization are determined by FSH and/or IGF-I. However, virtually nothing has been established concerning the action of this factor in ovarian cells. We investigated the effects of overexpressing either a wild-type (WT) Foxo1a or a dominant-negative truncation mutant of Foxo1a. We provide evidence that FoxO1a regulates the G1/S transition in this cell system, and that p27kip is posttranscriptionally regulated by FoxO1a. Our data indicate that FoxO1a both modulates p27kip abundance and directs its subcellular localization.

RESULTS A Dominant-Negative Foxo1a-⌬256 Increases the Expression of Proliferating Cell Nuclear Antigen (PCNA) We have previously demonstrated that growth factor and gonadotropin stimulation lead to the nuclear exclusion of FoxO1a in a PI3-kinase-dependent manner in our granulosa cell system (10). Because PI3-kinase signaling is important in cell cycle progression, we hypothesized that FoxO1a might regulate granulosa cell replication. To test this hypothesis, we first established the expression of a WT-Foxo1a, or alternatively, a dominant-negative construct (Foxo1a-⌬256, which retains its DNA binding domain but lacks a C-terminal transactivation domain). In Fig. 1, we demonstrate, by both Western blot and immunohistochemistry, that we achieve high levels of infection and expression for both of these adenoviral constructs. The immunohistochemistry also shows a significant difference in the compartmentalization of the expressed proteins. Under low-serum conditions, WT-Foxo1 is partially or exclusively in the cytosol in more than 85% of infected cells as shown by the lack of merge of fluorescein isothiocyanate (FITC) and Hoechst. In contrast, the dominant-negative Foxo1a construct is more concentrated in the nucleus in almost 90% of the cells. We then examined expression of PCNA, a G1/S-regulated cell proliferation marker. Our data indicate that inhibiting endogenous FoxO1a action by using the dominant-negative Foxo1a-⌬256 construct significantly increased proliferation of granulosa cells as judged by this marker 24 h post infection (Fig. 2).

Fig. 1. Adenoviral Constructs Expressing a WT-Foxo1a and a Dominant-Negative Foxo1a-⌬256 Truncation Mutant Are Expressed in Granulosa Cells Western blot analysis and immunofluorescence microscopy demonstrates expression of WT-Foxo1a and Foxo1a⌬256 constructs in granulosa cells. Whole cell lysates were prepared, or cells fixed, 24 h post transduction. Cells were infected with a null adenovirus, an adenovirus expressing murine HA-tagged WT-Foxo1a, or an adenovirus expressing an HA-tagged dominant-negative Foxo1a-⌬256. Exogenously expressed Foxo1a proteins were detected with an anti-HA antibody. The null adenovirus is not HA-tagged and cannot be directly visualized. Transduction efficiency for both WT-Foxo1a and Foxo1a-⌬256 infections was more than 75%. Expression of WT-Foxo1a and the truncated dominantnegative form were equivalent, however, under these conditions of 3% serum, overexpressed WT-Foxo1a was cytosolic in a large portion of cells whereas Foxo1a-⌬256 was predominantly nuclear.

FoxO1a Regulates the G1/S Transition in Granulosa Cells We next investigated the effect of introducing the WTFoxo1a or the dominant-negative Foxo1a-⌬256 construct on the cell cycle distribution in granulosa cells. DNA content as determined by propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS) analysis demonstrated that expression of these constructs altered the number of granulosa cells progressing through the G1/S phase transition (Fig.

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there was no demonstrable change in p27kip mRNA levels in these cells (Fig. 5B). These data suggest that FoxO1a regulates p27kip posttranscriptionally in this system. Expression of WT-p27kip with the DominantNegative Foxo1a-⌬256 Partially Restores the Normal Cell Cycle Distribution in Granulosa Cells

Fig. 2. A Dominant-Negative Foxo1a-⌬256 Increases the Expression of PCNA Shown is a bar chart demonstrating changes in PCNA, a G1/S-regulated protein, in cells uninfected or infected with a null virus, WT-Foxo1a, or Foxo1a-⌬256 (n ⫽ 5 experiments; *, P ⬍ 0.001). A representative Western blot is shown below.

3A). We found that although expression of WT-Foxo1a decreased entry into the cell cycle, expression of the dominant-negative Foxo1a-⌬256 caused a significant increase in the number of S-phase cells (61.2% vs. 7.7% of null-infected cells or 3.2% of WT-Foxo1infected cells) (Fig. 3B), suggesting that FoxO1a can regulate cell cycle entry. In agreement with the increase in DNA content by FACS analysis, cells transduced with Foxo1a-⌬256 also showed a significant increase in the number of S-phase cells detected by immunohistochemistry after bromodeoxyuridine (BrdU) incorporation (Fig. 4A). In Foxo1a-⌬256infected cells, 79.6% were labeled vs. 7.6% of nullinfected cells or 5.0% of WT-Foxo1-infected cells during a 4-h BrdU pulse (Fig. 4B). Collectively, these experiments demonstrate a dramatic increase in the number of cells actively synthesizing DNA when the dominant-negative Foxo1a was expressed. p27kip Protein, But Not mRNA, Is Increased after Expression of WT-Foxo1a in Granulosa Cells FOXO family members have been shown to induce expression of p27kip, a cdk inhibitor that plays a key role in cell cycle regulation (16, 20, 25, 32). We examined the expression of p27kip in granulosa cells expressing either WT-Foxo1a or the dominant-negative Foxo1a-⌬256 construct. There was a significant increase in p27kip protein in cells infected with WTFoxo1a and a decrease in p27kip when cells were infected with Foxo1a-⌬256 (Fig. 5A). Surprisingly,

Because levels of endogenous p27kip protein were increased in granulosa cells expressing the WTFoxo1a, and reduced in Foxo1a-⌬256-infected cells, we hypothesized that changes in this protein could account for the observed effects of FoxO1a on cell cycle progression. To address this issue, we infected granulosa cells with both the dominant-negative Foxo1a-⌬256 construct and an adenovirus expressing WT p27kip (Fig. 6). Significant overexpression of p27kip protein in granulosa cells coinfected with Foxo1a⌬256 reduced the cell cycle entry caused by expressing Foxo1a-⌬256 alone by approximately 50% (35.0% S-phase vs. 66.7% S-phase, respectively) (Fig. 6, A and B). These results suggest that p27kip is an important mediator of FoxO1a restraint on granulosa cell replication. However, regulation of p27kip levels is not the only mechanism by which FoxO1a inhibits cell cycle progression (or by which the dominant-negative Foxo1a increases DNA synthesis in these cells). Nuclear Localization of p27kip Is Inhibited in Granulosa Cells Expressing a Dominant-Negative Foxo1a Although we detected strong expression and hightransduction efficiency rates in preliminary studies with the adenoviral WT-p27kip, we questioned whether we were achieving effective coexpression of both WTp27kip and the Foxo1a constructs. Limited coexpression might explain the partial effect we observed by FACS in Fig. 6. To further examine the relationship between FoxO1a and p27kip, and to confirm coexpression in the same cells, we used immunohistochemistry to examine p27kip protein expression and subcellular localization in cells infected with both p27kip and each of the two Foxo1a constructs. In cells infected with the WT-p27kip virus alone, p27kip overexpression was detected in greater than 70% of cells, and the p27kip protein appeared to be mainly nuclear in location, as expected (data not shown). When we coinfected the cells with both WT-p27kip and WT-Foxo1a, we again found that p27kip was concentrated in the nucleus (Fig. 7A). In contrast, when we coinfected granulosa cells with WT-p27kip and Foxo1a-⌬256, we found that expression of the dominant-negative Foxo1a-⌬256 resulted in p27kip becoming largely localized to the cytosol, sometimes exclusively (Fig. 7A). Morphometric analysis of coinfected granulosa cells revealed that when WT-Foxo1a was expressed, p27kip was localized to the nucleus in 93% of coinfected cells vs.



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Fig. 3. FoxO1a Regulates the G1/S Transition in Granulosa Cells Shown is a representative flow cytometry histogram (A) of PI-stained cells, after infection with a null virus, WT-Foxo1aexpressing virus, or the dominant-negative Foxo1a-⌬256-expressing virus. DNA content analyses, as calculated by ModFit (hatched area under the curve), are summarized in the bar graph (B). In cells expressing Foxo1a-⌬256, we found a significant increase in S-phase cells (n ⫽ 5; P ⬍ 0.001), suggesting that FoxO1a can increase entry into the cell cycle.

approximately 25% when Foxo1a-⌬256 was coexpressed (Fig. 7B). Even more dramatic was the observation that p27kip was detected in the cytosol of fewer than 7% of cells coinfected with WT-Foxo1a. In con-

trast, greater than 75% of cells coinfected with Foxo1a-⌬256 had detectable levels of p27kip in the cytosol (25% of which were cells with p27kip predominantly or exclusively in the cytosol).



Fig. 4. FoxO1a Regulates the G1/S Transition in Granulosa Cells Immunohistochemistry (A) demonstrating an increase in BrdU incorporation in cells infected with a dominant-negative Foxo1a-⌬256 adenovirus vs. null adenovirus (4 h pulse, 20–24 h post transduction). When enumerated (B), there was a significant increase in BrdU incorporation in cells infected with dominant-negative Foxo1a (n ⫽ ⬃4000 nuclei; average 100 nuclei/10 random fields per construct; P ⬍ 0.001), indicating an increase in S-phase cells.

Overexpression of WT-p27kip Does Not Block the Increase in BrdU Incorporation Observed in Granulosa Cells Expressing a Dominant-Negative Foxo1a To determine more directly whether the localization of p27kip had a major impact on the replicative activity of granulosa cells, we coinfected cells with WT-p27kip and either a null virus or one of the two Foxo1a constructs. DNA synthesis was detected by BrdU incorporation. In this experiment, we were able to make the direct observation that p27kip was less active by virtue of its relocalization to the cytosol in these cells. In contrast, p27kip was predominantly nuclear in cells infected with the WT-Foxo1a or null virus. Among these coinfected cells, there were virtually none which were in S-phase (indicated by the absence of colocalization of p27kip and BrdU). In agreement with data from our FACS studies in which we examined cell cycle profiles of granulosa cells after coinfection with WT-p27kip, BrdU incorporation detected immunohis-

tochemically confirmed that normal rates of DNA synthesis were not restored in dominant-negative Foxo1a-⌬256-infected cells that were also infected with WT-p27kip (Fig. 8). Although p27kip was expressed at high levels, BrdU labeling continued to be significantly increased when FoxO1 function was inhibited.

DISCUSSION Our data show that FoxO1a plays a critical role in granulosa cell cycle control, in part due to its regulation of p27kip abundance and nuclear localization. Use of adenoviral vectors to overexpress WT-Foxo1a and a truncation mutant of Foxo1a that lacks its transactivation domain have allowed us to readily investigate FoxO1a functional endpoints to begin to determine the physiologic relevance of FoxO factors in granulosa cells. As previously demonstrated, FoxO1a is inacti-


Fig. 5. p27kip Protein, But Not mRNA, Is Increased after Expression of WT-Foxo1a in Granulosa Cells Shown in the top panel (A) is a bar chart demonstrating the increase in p27kip protein in cells infected with WT-Foxo1a vs. null, and a decrease in p27kip protein when granulosa cells were infected with the dominant-negative Foxo1a-⌬256 vs. null (n ⫽ 7 experiments; *, P ⬍ 0.001; **, P ⬍ 0.05). A representative Western blot is shown below. In the middle panel (B), RT-PCR products from RNA extracted from granulosa cells infected as above, reverse transcribed, and analyzed with primers for the p27kip gene or the ribosomal L19 gene, amplified as an internal control (29 cycles). There was no significant difference in mRNA between cells infected with WT-Foxo1a vs. null or Foxo1a-⌬256 (n ⫽ 3; ANOVA, P ⫽ 0.934).

vated by FSH and IGF-I via phosphorylation and nuclear exclusion. Accordingly, this pathway likely plays a major role in mediating follicular growth and/or survival under the influence of these factors. Forkhead transcription factors have been implicated in a remarkable number of diverse cellular processes from development and metabolism, to stress and aging, to proliferation and programmed cell death, depending on the family member and the system being studied (33, 34). Investigations focused on the FOXO subfamily have demonstrated that these transcription factors regulate the expression of a number of genes that are critical for the proliferative status of the cell as well as, in some tissues, genes involved in pro-


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grammed cell death (15, 16, 35). Because we could not predict whether overexpression of WT-Foxo1a or the dominant-negative Foxo1a would result in an altered growth and/or a survival phenotype, our first set of studies examined DNA content 24 h after infection with either of the adenoviral constructs. By FACS analysis, we did not find a major difference in apoptosis (n ⬍ 1) between the constructs at 24 h post transduction. However, caspase-3, a late marker of apoptosis, was activated by Western blot analysis in WT-Foxo1-infected cells (data not shown). The role of FoxO1a in granulosa cell death or survival must await more exacting studies. A more dramatic effect was observed in the cell cycle profiles obtained by FACS. We immediately observed a significant increase in the number of cells in S-phase (DNA between 1N and 2N as calculated by ModFit software) when the dominant-negative Foxo1a-⌬256 was expressed (Fig. 3). This was in agreement with the increase in PCNA (Fig. 2) and the increase in BrdU incorporation we found in these dominant negative-expressing granulosa cells (Fig. 4). Several reports have revealed that FOXO-induced cell cycle arrest either correlates with, or in one case is dependent upon, p27kip expression (16, 20, 22). Proliferation of mammalian cells is under strict control, and p27kip is an essential participant in this regulation both in vitro and in vivo, along with multiple other components. First identified as an inhibitor of cyclin E-cdk2, p27kip arrests the cell cycle in G0 or early G1 when overexpressed in cultured cells (36, 37). It is well established that p27kip levels are highest in quiescent cells and decline in response to growth factors or other mitogenic stimuli. The critical role of p27kip in regulation of proliferation is perhaps best illustrated in the p27kip knockout mouse, which exhibits gigantism due to increased cell number (38, 39). In addition, the female p27kip⫺/⫺ mice are sterile (38, 39). Thus, p27kip appears to play an important role in fertility of normal cycling animals, which exhibit an increase in p27kip in follicular cells when they cease growing after ovulation (31). Based on reports in the literature in other cell systems, we hypothesized that altered levels of p27kip expression might be mediating the changes in G1/S progression we observed in our granulosa cells as a result of infection with the Foxo1a adenoviral constructs. Subsequently, we determined that endogenous p27kip protein levels were indeed increased when the WT-Foxo1a was expressed, and decreased when Foxo1a-⌬256 was expressed, as predicted (Fig. 5A). In contrast to several other reports, we did not find altered mRNA levels of p27kip under our culture conditions (Fig. 5B), suggesting that transcriptional regulation of p27kip by FoxO1a did not account for the observed changes in cell cycle profiles, but rather was due to some posttranscriptional mechanism. Multiple posttranscriptional mechanisms are known to regulate p27kip activity, which is controlled not only by its concentration, but also by its distribution among



Fig. 6. Coexpression of WT-p27kip with the Dominant-Negative Foxo1a-⌬256 Partially Restores the Normal Cell Cycle Distribution in Granulosa Cells FACS analysis was performed as described in Fig. 3. Cells were infected with WT-p27kip in addition to either a null adenovirus, WT-Foxo1a, or the dominant-negative Foxo1a-⌬256. As shown in both the representative flow histograms (A) and the bar chart (B), overexpression of p27kip in the Foxo1a-⌬256-infected cells resulted in an approximately 50% decrease of S-phase cells vs. Foxo1a-⌬256 alone. Different superscripts represent statistically significant different results (n ⫽ 3; *, P ⬍ 0.001; **, P ⬍ 0.01).



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Fig. 7. Nuclear Localization of p27kip Is Inhibited in Granulosa Cells Expressing a Dominant-Negative Foxo1a. Granulosa cells were infected with an adenovirus expressing WT-p27kip and coinfected with a null adenovirus (data not shown), WT-Foxo1a (HA-tagged), or Foxo1a-⌬256 (HA-tagged). Shown is immunofluorescence microscopy from a representative experiment (A) demonstrating that p27kip is relocalized to the cytosol either partially (small arrows) or exclusively (large arrow) in cells coexpressing a dominant-negative Foxo1a. Cells that expressed both p27kip and one of the Foxo1a constructs were enumerated and scored for p27kip localization within the cell (B) (*, predominantly; **, equally; 30–100 coinfected cells were scored in each of five random fields per coinfection group; ⬖n ⬇ 650 cells; P ⬍ 0.001).

different cellular complexes, and its subcellular localization (37). One of the key mechanisms involved in controlling levels of p27kip in cycling cells is targeted degradation by the ubiquitin-proteosome system (26, 40). This pathway may play a role in the Foxo1a-⌬256dependent decrease in p27kip in granulosa cells because cells expressing the dominant-negative Foxo1a⌬256 exhibit a decrease in p27kip that is partially reversed by treatment of the cells with a general proteasome inhibitor, MG132 (data not shown). This would indicate a role for FoxO1a in stabilizing p27kip by counteracting proteosome-mediated proteolysis in some way, although this mechanism remains to be elucidated. This pathway (which is well supported in the literature) will be investigated in more detail in future studies. Our initial studies (Figs. 2–4) suggested that levels of p27kip correlated inversely with S-phase entry induced by experimental changes in FoxO1a activity. To exam-

ine the role of p27kip more directly, we overexpressed this protein in cells expressing the dominant-negative Foxo1-⌬256. Overexpressing WT-p27kip inhibited entry into the cell cycle but it only reduced the effect of Foxo1-⌬256 on granulosa cell S-phase entry by about 50% (Fig. 6). Immunohistochemical experiments allowed us to verify that these cells coexpressed both constructs. In addition, these studies showed dramatic differences in p27kip localization within the cell. Blocking FoxO1a resulted in decreased nuclear p27kip. Because nuclear localization is a prerequisite for p27kip to function as a cell cycle regulator, our data suggest a novel FoxO1-mediated regulatory mechanism of p27kip, whereby its inhibitory properties are functionally activated or maintained. Recently, data in other systems have also indicated that compartmentalization of p27kip during cell cycle progression plays a critical role in the actions and



Fig. 8. Overexpression of WT-p27kip Does Not Block the Increase in BrdU Incorporation Observed in Granulosa Cells Expressing the Dominant-Negative Foxo1a-⌬256 Granulosa cells were coinfected with WT-p27kip and either the null, WT-Foxo1a, or Foxo1a-⌬256 constructs as in Fig. 7, and were additionally pulsed with BrdU for 4 h before fixation. Cells were stained for p27kip expression (FITC labeled) and for BrdU incorporation (Texas Red labeled). In agreement with the flow cytometry data presented in Fig. 6, BrdU incorporation is not reduced by the coexpression of p27kip in the Foxo1a-⌬256-infected cells.

regulation of this protein. Multiple reports have shown that p27kip is almost completely nuclear in G0, whereas mitogenic stimulation causes cytoplasmic redistribution (26, 41, 42). It has been suggested that phosphorylation of p27kip on Ser10 and Thr157, by specific kinases such as kinase-interacting stathmin and Akt, are largely responsible for this compartmentalization (27–30). Our data suggest that FoxO1a also modulates p27kip subcellular localization, although the mechanism of this effect is unknown. FoxO1a may induce, or indirectly repress, the expression of an intermediate phosphatase or kinase, respectively, which then regulates the phosphorylation and subcellular localization of p27kip. When FoxO1a is inhibited, as with FSH (9, 10) or a dominant-negative Foxo1a construct, the abundance of p27kip in the nucleus may fall below the threshold required for activation of cyclin-cdk2 complexes (30, 42, 43). This concept is supported by the fact that G1 nuclear export of p27kip precedes both cdk2 activation and degradation of the bulk of p27kip (43). In the cytosol, p27kip likely fails to inhibit events involved in the G1/S transition and may be susceptible to other regulatory processes that affect its stability/ degradation. In the ovary, data have suggested that granulosa cell proliferation during follicular development is regulated, at least in part, by gonadotropins and estradiol that increase levels of cyclin D2 (the major cyclin D isoform in granulosa cells) relative to levels of p27kip (31). As discussed above, this relative difference may be achieved by altering the abundance or the localization of p27kip. In our previous study, we demonstrated that IGF-I and FSH regulate FoxO1a compartmentalization, and thus activity (10). p27kip also appears to be regulated by its compartmentalization. One explanation for these data could be that FoxO1a acts as a chaperone for p27kip, binding and escorting it out of the nucleus. However, to date, we have not

found a protein-protein interaction between FoxO1a and p27kip by coimmunoprecipitation studies (data not shown). This report demonstrates for the first time that FoxO1a plays a role in regulation of p27kip and the cell cycle in granulosa cells. Although much remains to be learned about these phenomena, this overexpression strategy has been useful to provide a first functional analysis of FoxO1a in granulosa cells and has shed new light on cell cycle regulation by these factors. To combat misinterpretation due to significant overexpression of these constructs, it will be necessary to complement these studies with others examining endogenous FoxO1a to confirm these findings. The expression and regulation of FoxO1a (and the genes and proteins it regulates, in turn) are extremely relevant to the ovary where they may govern granulosa cell replication and follicle growth.

MATERIALS AND METHODS Cell Culture and Viral Infection Porcine ovarian granulosa cells were harvested from medium-sized antral follicles (5–10 mm) as previously described (44) and used for all experiments. Briefly, granulosa cells were scraped from follicle walls, resuspended in plating medium containing DMEM and F-10 medium (1:1), 1% penicillinstreptomycin, gentamicin (0.5 ␮g/ml) and amphotericin B (0.5 ␮g/ml), supplemented with 10% fetal bovine serum (FBS), transferrin (5 ␮g/ml), insulin (3 U/ml), and FSH (0.5 ng/ml). The cells were then grown to confluence before trypsinization and freezing. After this single passage, granulosa cells were plated in medium (as above) supplemented with 10% FBS for 48–72 h (subconfluent) with one medium change (24 h before infection period). Serum and other culture supplies were from Invitrogen Life Technologies (Carlsbad, CA). For adenoviral infection, cells were washed once in PBS and infected with a null adenovirus, a WT-Foxo1a-expressing adenovirus, or a dominant-negative mutant Foxo1a-⌬256. In certain experi-


ments, cells were coinfected with a WT-p27kip-expressing adenovirus. Infection was carried out at a multiplicity of infection (MOI) ⫽ 10 in all cases. The Foxo1a adenoviral constructs were gifts from D. Accili (College of Physicians and Surgeons, Columbia University, New York, NY) and were constructed as described in Ref. 45. The WT-p27kip adenovirus was the gift of K. H. Park (Vanderbilt University, Nashville, TN) and C. T. Lee (Seoul National University, College of Medicine, Seoul, Korea), and was constructed as described in (46). These viruses were amplified in monolayer cultures of 293 cells and plaque assays were performed to determine titers of virus in crude cell lysates, using standard methods as described in (47, 48). Granulosa cells were infected with lysates during a 1.5-h adsorption period in serum-free medium that was subsequently replaced with 3% FBS medium for 24 h. Antibodies and Reagents The following primary antibodies were used for this study: rabbit anti-HA (hemagglutin), mouse anti-HA, rabbit antip27kip (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-BrdU (Oncogene Research Products/Calbiochem, San Diego, CA), and mouse antiactin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies include: antirabbit IgG-horseradish peroxidase and antimouse IgG-horseradish peroxidase (Santa Cruz Biotechnology), Texas Red-conjugated antimouse Ig(H&L), FITC-conjugated antirabbit Ig(H&L), and FITC-conjugated antimouse Ig(H&L) (Southern Biotechnology Associates, Inc., Birmingham, AL). All other reagents were from Sigma-Aldrich. Immunoblot Analysis Cells were harvested 24 h after infection. Whole cell lysates were prepared by scraping granulosa cells in 250 ␮l of cold lysis buffer [20 mM HEPES (pH 7.9), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1.0% Nonidet P-40] with additional protease and phosphatase inhibitors (2 ␮g/␮l leupeptin, 2 ␮g/ul aprotinin, 2 ␮g/␮l pepstatin, 0.2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM sodium fluoride) and 0.5 mM dithiothreitol. Protein concentrations were determined by a protein assay (Bio-Rad, Hercules, CA). SDS-PAGE sample buffer was added and the samples were heat denatured. Proteins were separated by SDS-PAGE (4–12% gradient acrylamide running gel), and transferred to nitrocellulose. The membranes were blocked in 5% nonfat milk in TBS-T [10 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] for 1 h at room temperature, and probed with primary antibody (1:1000) for 1.5 h at room temperature (or overnight at 4 C). After a series of 5-min TBS-T washes, the blot was incubated with a secondary antibody (1:5000) for 1 h at room temperature. The membrane was washed two times (20 min) in TBS-T and antigen-antibody complexes were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia, Piscataway, NJ) exposed on Kodak Biomax Light film (Rochester, NY). Quantitation of bands was performed using a Molecular Dynamics densitometer (Sunnyvale, CA) and Bio-Rad Quantity One software. RT-PCR Total RNA was extracted from granulosa cells using the RNeasy RNA isolation kit (QIAGEN, Valencia, CA) according to the manufacturer. Reverse transcription was carried out using Superscript II (Invitrogen Life Technologies). Briefly, 2 ␮g of total RNA were mixed with 1 ␮M oligo-deoxythymidine16 primer (Roche/Applied Biosystems, Foster City, CA) and incubated at 70 C for 10 min. Reactions were placed on ice for 5 min and mixed with dithiothreitol, deoxynucleotide triphosphates, 5⫻ reverse transcriptase buffer, and incu-


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bated at 42 C for 5 min. Superscript II ribonuclease H-reverse transcriptase was then added and the reaction was carried out at 42 C for 1 h and terminated by incubating at 70 C for 15 min. The following oligonucleotides were synthesized for use as PCR primers: 5⬘-ATAAAGTCCTTCCCGCTGAC-3⬘ (p27kip sense); 5⬘-GGCTCCTTAGAAACTCCCTTG-3⬘ (p27kip antisense); 5⬘-AAGCCTGTGACTGTCCATTCC-3⬘ (L19 sense); 5⬘-TGCTCCATGAGAATCCGCTTG-3⬘ (L19 antisense). After activating Taq for 3 min at 95 C, PCR was carried out for 29 cycles (denaturing at 94 C for 30 sec, annealing at 58 C for 30 sec, and extension at 72 C for 45 sec) in a 50-␮l reaction containing cDNA, 100 nM of each gene-specific primer, 10⫻ PCR buffer, deoxynucleotide triphosphate, and JumpStart Taq Polymerase (Sigma-Aldrich). The PCR products were electrophoresed on a 1.2% agarose gel. Preliminary experiments with varying cycle number and/or varying template concentrations showed the result depicted to be on the linear portion of the amplification curve. Cell Cycle Analysis DNA content of infected granulosa cells was determined by PI staining of DNA and FACS analysis. Cells were collected and washed in PBS before being fixed in ice-cold 70% ethanol overnight. Cells were then centrifuged (800 rpm for 10 min) and the supernatant was removed. Cells were resuspended in Vindelov’s reagent [1 M Tris-buffered saline (pH 7.6), ribonuclease A (10 ␮g/ml), PI (50 ␮g/ml), and 0.1% Nonidet P-40] and incubated for 1.5 h at room temperature. A 40-␮m mesh was used to filter cells before being analyzed on the FACScan (Becton-Dickinson, Franklin Lakes, NJ). Data analysis was performed using ModFit LT software (Verity Software House, Inc., Topsham, ME). Immunocytochemical Analysis Granulosa cells were plated on glass chamber slides (Nalge Nunc International, Naperville, IL) and grown as above. They were infected with one or more adenoviral constructs for 1.5 h and maintained in 3% FBS for 24 h. For BrdU incorporation experiments, BrdU (10 ␮M) was added directly to the culture medium at 20 h post infection (4-h pulse). Cells were then washed twice in PBS and fixed in 4% paraformaldehyde for 10 min. After two 5-min washes in PBS, the cells were permeabilized in acetone/methanol (1:1) for 1 min and washed twice more in PBS. The fixed cells were then blocked in 1% normal goat serum for 1 h and incubated with primary antibody overnight at 4 C. After three 10-min washes in PBS, the cells were incubated with a goat secondary antibody for 2 h and Hoechst 33342 (Molecular Probes, Eugene, OR) for 15 min. Visualization and morphometric analysis took place on an IX50 inverted system microscope (Olympus, Tokyo, Japan) using SPOT RT software (Diagnostic Instruments, Inc., Sterling Heights, MI). Statistics Each result depicted reflects data from at least three independent experiments. For critical points, multiple independent experiments were quantified and analyzed by ANOVA with a Tukey’s post hoc test (GraphPad Prism software, San Diego, CA).

Acknowledgments We are extremely grateful to Domenico Accili (College of Physicians and Surgeons, Columbia University, New York, NY) for providing the adenoviral constructs WT-Foxo1a and Foxo1a-⌬256, and Paul Park (Vanderbilt University, Nash-



ville, TN) and Choon-Taek Lee (Seoul National University, College of Medicine, Seoul Korea) for Ad-WT-p27kip. We thank David Spector for technical advice, and Michael Verderame and Patrick Quinn (all from Penn State University, College of Medicine, Hershey, PA) for comments on the manuscript.

Received February 18, 2004. Accepted April 6, 2004. Address all correspondence and requests for reprints to: James Hammond, The Pennsylvania State University, College of Medicine, 500 University Drive, C6636, Hershey, Pennsylvania 17033. E-mail: [email protected] This work was supported in part by National Institutes of Health Grant HD-24565.

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